Biophysical chemistry behind sickle cell anemia and the mechanism of voxelotor action

Sickle cell anemia disease has been a great challenge to the world in the present situation. It occurs only due to the polymerization of sickle hemoglobin (HbS) having Pro–Val–Glu typed mutation, while the polymerization does not occur in normal hemoglobin (HbA) having Pro–Glu–Glu peptides. It is also well confirmed that the oxygenated HbS (OHbS) does not participate in the polymerization, while the deoxygenated HbS (dHbS) does, which causes the shape of red blood cells sickled. After polymerization, the blood has a low oxygen affinity. Keeping this fact into consideration, only those drugs are being synthesized that stabilize the OHbS structure so that the polymerization of HbS can be stopped. The literature data showed no systematic description of the changes occurring during the OHbS conversion to dHbS before polymerization. Hence, an innovative reasonable study between HbA and HbS, when they convert into their deoxygenated forms, was done computationally. In this evaluation, physiochemical parameters in HbA/HbS before and after deoxygenation were studied and compared deeply. The computationally collected data was used to understand the abnormal behaviour of dHbS arising due to the replacement of Glu6 with Val6. Consequently, during the presented computational study, the changes occurring in HbS were found opposite/abnormal as compared to HbA after the deoxygenation of both. The mechanism of Voxelotor (GBT-440) action to stop the HbS polymerization was also explained with the help of computationally collected data. Besides, a comparative study between GBT-440 and another suggested drug was also done to know their antisickling strength. Additionally, the effect of pH, CO, CO2, and 2,3-diphosphoglycerate (2,3-DPG) on HbS structure was also studied computationally.


Mohd. Suhail
Sickle cell anemia disease has been a great challenge to the world in the present situation.It occurs only due to the polymerization of sickle hemoglobin (HbS) having Pro-Val-Glu typed mutation, while the polymerization does not occur in normal hemoglobin (HbA) having Pro-Glu-Glu peptides.It is also well confirmed that the oxygenated HbS (OHbS) does not participate in the polymerization, while the deoxygenated HbS (dHbS) does, which causes the shape of red blood cells sickled.After polymerization, the blood has a low oxygen affinity.Keeping this fact into consideration, only those drugs are being synthesized that stabilize the OHbS structure so that the polymerization of HbS can be stopped.The literature data showed no systematic description of the changes occurring during the OHbS conversion to dHbS before polymerization.Hence, an innovative reasonable study between HbA and HbS, when they convert into their deoxygenated forms, was done computationally.In this evaluation, physiochemical parameters in HbA/HbS before and after deoxygenation were studied and compared deeply.The computationally collected data was used to understand the abnormal behaviour of dHbS arising due to the replacement of Glu6 with Val6.Consequently, during the presented computational study, the changes occurring in HbS were found opposite/abnormal as compared to HbA after the deoxygenation of both.The mechanism of Voxelotor (GBT-440) action to stop the HbS polymerization was also explained with the help of computationally collected data.Besides, a comparative study between GBT-440 and another suggested drug was also done to know their antisickling strength.Additionally, the effect of pH, CO, CO 2 , and 2,3-diphosphoglycerate (2,3-DPG) on HbS structure was also studied computationally.
Sickle cell anemia disease (SCAD) is a molecular disease 1 that occurs due to the gene mutation 2 .The low oxygen affinity of a mutant hemoglobin is a characteristic of SCAD 3,4 .It has already been confirmed 2 that one of the glutamic acid residues of Pro-Glu-Glu (PGG) present in normal hemoglobin (HbA) is replaced by valine residue i.e.Pro-Val-Glu (PVG) in sickle cells, also called E6V mutation.Although sickle hemoglobin (HbS) has normal oxygen binding affinity, its polymerized form does not 5 .The production of abnormal Red Blood Cells (RBCs) under hypoxic conditions [4][5][6][7][8] and low HbS solubility 9,10 are just because of the polymerization of the same [5][6][7][8][9][10] .Actually, the deoxygenated form of HbS (dHbS) takes part in the polymerization reaction rapidly while its oxygenated form (OHbS) does not 7 .Keeping this fact into consideration, the OHbS is made pharmacologically more stable so that the HbS polymerization can be inhibited to prevent RBC sickling and provide long-term disease modification [11][12][13] .It must be remembered that the polymerization of a biomolecule involves two steps.The first is the conformational change of the native monomer; the second is the binding of the 'open' , flipped, monomer to the growing interdigitating polymer 14 .Although different researchers have given different perspectives associated with SCAD and the polymerization of sickle hemoglobin, they did not give a deep insight which is why their studies raise many questions.Different aspects in HbA/HbS evaluation with their gaps are as follows:

Biophysical interaction-based evaluation
The steric and electrostatic effects of the residue hosted through the mutation 15 have also been found as 7,[15][16][17] a reason for the low oxygen affinity of a mutant hemoglobin.As per the fundamental electrostatic analysis 16,17 , it has been confirmed that the effect/involvement of E6V mutation in the polymerization of dHbS is not direct.This is because Glu6 itself does not seem to be engaged in any significant electrostatic interaction with Hb's interacting partners 18 .Hence, the loss of Glu6's side chain leads to indirect local electrostatic equilibrium perturbation as per intermolecular electrostatic interactions.These intermolecular interactions include salt bridges, hydrogen bonds, and charge-dipole interaction which may stabilize [19][20][21] or destabilize 22,23 biomolecules [24][25][26] .This electrostatic equilibrium perturbation is of functional significance for the overall structural stability of Hb 17 .Moreover, the presence and strength of the salt bridge make the Hb molecule stable which was proved when a different form of HbS 27 was observed after the substitution of Lys132 with Asn132.It is because Lys132 forms a Lys132-Glu7 typed salt bridge with Glu7 but no salt bridge is formed between Asn132 and Glu7 because Asn132 carries no charge.Therefore, the interruption in the salt bridge caused by the Asn-Lys substitution directly affected the α 2 β 2 stability, underlining the disquiet of local electrostatic equilibrium by SCD-linked mutations such as Lys132Asn and E6V too.The same thing was also supported by the earlier study of molecular dynamics simulation confirming the inter-facial electrostatic collaborations accountable for HbS polymerization 7,28 .Literature data shows the lack of comparative study between OHbS and dHbS including the variation occurring in biophysical interactions along with the OHbS conversion into dHbS before polymerization.Besides, the effect of the existence of not only 2,3-diphosphoglycerate (2,3-DPG) but also the anti-sickling drugs (GBT440) on biophysical interactions in HbS was found out of expectations.Keeping these facts into consideration, the biophysical interaction-based mechanism of GBT440 action was explained in the presented article.Besides, the same article also gives the biophysical interaction-based explanation of how this sickle anemia-linked E6V mutation causes the sickling of RBC via dHbS polymeirzation.

Nature-based evaluation
Quantum level study has clearly shown that the PVG has a more reactive nature to take part in biochemical reactions as compared to PGG 29 .Moreover, the normal Pro-Glu-Glu peptides are hydrophilic in nature, while the mutant Pro-Val-Glu peptides are hydrophobic due to the presence of hydrophobic valine residue [30][31][32] .Hence, researchers [30][31][32] have also suggested that the hydrophobic R-group of Val-beta6 (E6V) appears to insert into a hydrophobic pocket constituted by β88 leucine (Leu-beta88), β85phenylalanine (Phe-beta85), and β73 aspartic acid (Asp-beta73) residues on adjacent deoxygenated sickle hemoglobin molecules (dHbS-M) 33,34 .For the insertion of the R-group of Val-beta6 in the acceptor pocket regions on adjacent dHbS-M, not only stereospecificity to the Leu-beta88 side chain 30 but also the contact position on dHbS-M was found favourable [30][31][32][33][34] .On the other hand, no such favourable conditions were noted for the insertion of the hydrophilic R-group of Glu-beta6 (β-glutamic acid) into the same which is why polymerization does not occur in deoxyHbA 35 .This thing does not give any correlation with electrostatic forces present in HbS/HbA.It is because the nonpolar suppressed surface area reflects many things such as the hydrophobic effect, the major driving potency in protein binding and folding, but to flexible extents 36,37 .Besides, PVG and PGG both are present on the surface, and both can make salt bridges for hemoglobin stability because the salt bridges present on the surface provide stability to protein 38 .The presented article explains why a hydrophobic residues i.e.PVG inserts into the neighboring hydrophobic pockets despite providing stability to a protein via salt bridge formation.

Medium-based evaluation
Like the studies described above, the effect of an acidic/basic medium on dHbS polymerization has great importance whose appropriate and deep explanation still needs confirmation.Different studies were done to understand the effect of pH, carbon monoxide, and other factors on HbS polymerization.For example, the main findings regarding the pH effect showed that the lowered pH over the physiological range promotes not only HbS polymerization but also RBC sickling 39 .Hence, the chance for polymerization of HbS is increased at lower pH.The exact mechanism by which protons (H + ) facilitate the HbS polymerization to cause a lowering in oxygen affinity at low pH is not known 39 .Moreover, the decrease in hemoglobin affinity for oxygen caused by the accumulation of 2,3-DPG (2,3-diphosphoglycerate) has also been found to correlate with pH 10,40 .This mechanism for the intracellular accumulation of 2,3-DPG (2,3-diphosphoglycerate) increases oxygen release from RBCs under circumstances where it is needed most 41 .This release is potentiated by the Bohr Effect, in which hemoglobin's binding affinity for oxygen is also reduced because of the high concentration of carbon dioxide.In tissues with high energetic demands, oxygen is rapidly consumed, which increases the concentration of H + and carbon dioxide 42 .It is well confirmed that the probability of RBCs sickling is also increased due to the higher intracellular concentration of 2,3-DPG in RBC hemoglobin 43,44 .Of course, the deoxygenated form of HbS takes part in polymerization reaction rapidly, but it must be remembred that the oxygen affinity and hemoglobin polymerization both are different things.It is because the loss of oxygen or less oxygen affinity is related to the entry of 2,3-DPG in hemoglobin structure [45][46][47] , while the polymerization reaction is related to the RBC sickling caused by residual combination in hemoglobin 48,49 .The most important thing noted in the literature data was the lower oxygen affinity of hemoglobin caused by the accumulation of 2,3-DPG at higher pH 10 , while at lower pH, the RBC sickling was found promoted 39 .The 2,3-DPG entry in RBCs can occur easily if the intracellular medium is slightly basic 10,50 which is why its entry is found pH dependent.The presented computationally-evaluated literature data explains how a decrease in intracellular pH is responsible for (i) the removal of 2,3-DPG from CO 2 -added medium, and (ii) the promotion of HbS polymerization.Besides these facts, (i) the HbS polymerization caused by the 2,3-DPG accumulation at higher pH, and (ii) the reason for hemoglobin stability caused by the addition of carbon monoxide gas are also explained deeply.

Anti-sickling drugs and their function
Voxelotor (GBT440), the first hemoglobin oxygen-affinity modulator 51 , when bound to HbS, inhibits polymerization and red blood cell sickling 52 .Therefore, the U.S. FDA has approved GBT440 (Fig. 1) as an anti-sickling agent 53 .The literature data related to the changes in HbS biophysical interactions caused by GBT440 (voxelotor) was not found yet.Hence, a satisfactory explanation of the mechanism of GBT440 action is given here after

Physiochemical parameters
Although there were many physiochemical interactions among polypeptide chains of HbA and HbS, only those physiochemical interactions were studied deeply which were linked to PGG and PVG residues.The stability at the mutation point of B, D, F, and H chains of dHbS was evaluated by comparing it with the same chains of OHbA/dHbA.It was observed that the PVG residues present in 'F and H' β-chains of dHbS were found as stable as those of OHbA.This is because the distance of electrostatic interactions made by PVG residues in 'F and H' β-chains of dHbS was found similar to the PGG residues of the same OHbA β-chains (Table 1 and Table 2).Moreover, the salt bridge number and their strength determine the stability of Hb protein as per section "Biophysical interaction-based evaluation" of the presented article.Hence, after deoxygenation, the PVG residues in 'F and H' β-chains of HbS attain the same stability as PGG do in OHbA i.e. no major changes occur in both chains of HbS after deoxygenation.
On the opposite side, the PVG residues were found not to provide the stability to 'B and D' β-chains of dHbS as PGG does to OHbA/dHbA.This is because the distance of the electrostatic interactions made by PVG residues in 'B and D' β-chains of dHbS was found very different from the PGG residues of the same dHbA β-chains (Table 1 and Table 2).Moreover, other structural differences between HbA and HbS were also found at the dissimilar points (PGG and PVG) of the 'B and D' β-chains of HbA and HbS.Therefore, keeping these facts into consideration, the biophysical interactions made by PGG and PVG were compared only in 'B and D' β-chains of HbA and HbS respectively.The number of salt bridges made by PGG residues of β-chain (B chain) was found 1 in both OHbA and dHbA with a distance of 3.57 A° and 2.91 A° respectively (Table 1).On the other hand, the number of salt bridges made by PVG residues of β-chain (B chain) was 1 in OHbS and 2 in dHbS with the distance of 2.63 A° and 3.18 A° & 3.22 A° respectively (Table 2).Furthermore, 1 salt bridge made by PGG residues of β-chain (D chain) was found in dHbA with a distance of 2.89 A° (Table 2), while no salt bridge was found at the mutation point (PGG) of the same dHbS chain (Table 2).
Besides the salt bridge, other types of interactions involved in Pro-Glu-Glu and Pro-Val-Glu residues in 'B and D' β-chains of HbA and HbS are also given in Table 1 and Table 2 respectively.It was found that the number of hydrogen bonds and their distance changed when OHbA/OHbS structures were compared with dHbA/dHbS (Table 1 and Table 2).On the other hand, the distinguished and extra salt bridge (B:LYS82:NZ-D:HIS146:O) was found (Table 2) in the β-chain of OHbS structure containing the anti-sickling drug, GBT440 (PDB ID: 5e83 13 ).In this additional salt bridge, the 'Lys residue' present in ASN-LEU-LYS-GLY-THR peptides of OHbS β-chain i.e. 'B' chain (5e83 13 ) was found attached to the 'HIS residue' of the adjacent β-chain (D chain).Such type of salt bridge was not found in the same β-chains of OHbA, dHbA, OHbS, and dHbS (pdb codes: 1hho 67 , 1a3n 68 , www.nature.com/scientificreports/5e6e 69 and 2hbs 70 respectively).Moreover, this type of additional salt bridge was also not found in VZHE-039 containing HbS protein (6xd9 53 ).In the case of VZHE-039 treated HbS, the distance of one of the two salt bridges in PVG residues was found the same but that of another one was found longer (Table 2) when compared with GBT-440 treated HbS (5e83 13 ).Moreover, the number of salt bridges on β-chain (B chain) was found greater in the hemoglobin (Hb) oxygenated with CO (2m6z 71 ), as compared to the hemoglobin oxygenated with oxygen (1hho 67 ) (Table 1).

Peptide bond torsion
The variation in peptide bond torsion also gave very significant points.A smooth variation in the peptide bond torsion was noted when all atoms present in PVG were tagged (Fig. 2) for the same (Tables 3, 4).It was noticed that the peptide bond torsion in Pro-Glu and Glu-Glu increases when oxygenated HbA converts into its deoxygenated form (Table 4).On the other hand, the peptide bond torsion in Pro-Val increases but that in Val-Glu decreases when oxygenated HbS (OHbS) converts into its deoxygenated form (dHbS).All these things were also noted (Table 4) in the case of TOHbS (oxygenated HbS treated with GBT440) so that the mechanism of action of the anti-sickling drug could be explained.Moreover, in the main findings observed during the structural evaluation of OHbA, dHbA, OHbS, and dHbS, the Ramachandran plot-based study of both residues (Pro-Glu-Glu and Pro-Val-Glu) was also found very supportive (Fig. 3a-d).Based on the Ramachandran plot study, when OHbA converts into dHbA, the value of Ф (phi) bond is decreased for Pro5, and that of Ѱ (psi) for the same increased, while only minor changes occur in values of Ф (phi) and Ѱ (psi) bonds for Glu6 and Glu7 (Fig. 3a,b).
Based on the same study, when HbS converts into dHbS, the value of Ѱ (psi) bond is increased for Glu7, and that of Ф (phi) for the same includes very little change, while no major changes occur in values of Ф (phi) and Ѱ (psi) bonds for Pro5 and Val6 (Fig. 3c,d).

The pH effect study and HbS interactions with 2,3-DPG
The pH effect on dHbS polymerization also gave tremendous facts associated with the control or acceleration of polymerization.When the protonation of the salt bridge-forming residues (Glu7 and Lys132) present on the β-chain of dHbS was done computationally, the salt bridge distance between them was found lesser as compared to their deprotonated forms.Therefore, the effect of protonation and deprotonation on the salt bridge was found directly related to the salt bridge distance.It was also found that the functional group of only Lys132 got www.nature.com/scientificreports/protonated, while that of Glu7 did not, after the computational protonation.Furthermore, when the structure of the HbS β-chain (1b86 72 ) was evaluated to know the effect of 2,3-DPG entry, an additional salt bridge between 2,3-DGP and the 'Lys residue' of ASN-LEU-LYS-GLY-THR peptides was noted.

Discussion
The results interpretation in the presented work removed the curtain from not only the reason for dHbS polymerization but also the mechanism of GBT440 action.In the results of physiochemical parameters among biological molecules (OHbA, dHbA, OHbS, and dHbS), the Gly7-Lys132 salt bridge distance on β-chain (B chain) was found greater in OHbA as compared to dHbA (Tables 1, 2).The short-ranged Glu7-Lys132 salt bridge in dHbA is an indication of its stability which makes the local environment rigid.This is because the stabilizing salt bridges make local regions inflexible for better fit due to functional requirements 20 .The formation of this more stable and short-ranged salt bridge can be related to the change in Pro5-Glu6 peptide bond torsion (Table 4).It is because, the conversion of OHbA to dHbA involves the change in Pro5-Glu6 peptide bond torsion to a greater extent i.e. from − 174.33 to + 174.14, while it occurs only to some extent in Glu6-Glu7 peptide bond i.e. from + 170.93 to + 179.45 (Fig. 4a).Actually, this torsional change occurs due to the motion of Pro5 residue around the Pro5-Glu6 peptide bond as per the Ramachandran plot (Fig. 3a,b).It is because the value of Ф (phi) bond is decreased for Pro5, and that of Ѱ (psi) for the same increased, while only minor changes are observed in values of Ф (phi) and Ѱ (psi) bonds for Glu6-Glu7 (Fig. 3a,b).Hence, during the torsional change in the Pro5-Glu6 peptide bond along with the OHbA conversion to dHbA, the Pro5 motion in the Pro5-Glu6 peptide bond creates a torsion due to which Glu6 moves in the opposite direction with respect to Glu7 (Fig. 4a).In this way, the www.nature.com/scientificreports/movement of Glu6 in opposite direction minimizes the created torsion.Besides minimization, the motion of Glu6 in the opposite direction concerning Glu7 also reduces the repulsion between two negatively charged species i.e.Glu6 and Glu7.Because of feeling less repulsion, Glu7 becomes able to make a more stable short-ranged salt bridge with Lys 132 after the conversion of OHbA to dHbA (Fig. 4a,b).In this way, the PGG residues in dHbA get stabilized due to this short-ranged and stronger salt bridge.
On the other hand, the salt bridge distance on the β-chain (B chain) was found lesser in OHbS as compared to dHbS.The long-ranged Glu7-Lys132 salt bridge in dHbS is the indication of its instability due to which the peptide chain feels conformational specificity for the fold or function which is provided by a destabilizing salt bridge 23 .Moreover, the increase in salt bridge distance in the case of dHbS is not only an indication of a weaker salt bridge but also responsible for the conversion of PVG into a movable/moving domain.This is because short-range electrostatic interactions are largely absent between moving domains 73 .Therefore, the Pro-Val-Glu residues gain flexibility in dHbS.Another reason behind the flexibility of Pro-Val-Glu is the hydrophobic nature of the 'valine' residue present in Pro-Val-Glu of HbS.It is because not only the weaker electrostatic interactions (salt bridge) but also the hydrophobicity allows conformational flexibilities in a protein 14,73 as shown in Fig. 4c.Moreover, the torsional change in Pro-Val-Glu peptide bonds along with the OHbS conversion to dHbS can not be ignored.The observation of the negatively charged Glu7 motion away from the charged Lys132 residue was done with the inclusion of a Ramachandran plot-based study (Fig. 3c,d).Besides, it also played an important role in the evaluation of all the things mentioned in the introductory part of the presented article.Actually, the conversion of OHbS to dHbS involves the change in Pro5-Val6 peptide bond torsion to a greater extent i.e. from − 178.33 to + 178.14 (Fig. 4a) which was found more as compared to Pro5-Glu6 in HbA.It is just because of the less hindered Pro5-Val6 peptide bond which makes Pro5-Val6 more movable around the peptide bond in HbS as compared to Pro5-Glu6 in HbA.Hence, the motion in Pro5-Val6 residues to a greater extent along with the OHbS conversion to dHbS creates a torsion on the Val6-Glu7 peptide which is minimized by the Glu7 motion.For this minimization, the Glu7 residue of HbS moves in that direction where the effect of torsion created by Pro5-Val6 peptide on Val6-Glu7 peptide could be minimal.This notable point regarding the Glu7 motion was also supported by the Ramachandran plot-based study (Fig. 3c,d) in which, the value of Ѱ (psi) bond for Glu7 was found to increase, and that of Ф (phi) for the same was found with no major change, while only minor changes were observed in values of Ф (phi) and Ѱ (psi) bonds for Pro5 and Val6.Subsequently, due to the occurrence of Val6-Glu7 peptide motion in the opposite way with respect to Pro5-Val6 peptide (Fig. 4a), a less stable salt bridge with a long distance is made between Glu7 and Lys132 after the OHbS conversion to dHbS (Fig. 4b).Thus, the PVG residues in the deoxygenated form of HbS get destabilized due to the long-ranged and weaker salt bridge.In this way, it clearly clarifies how the Val6 in place of Glu6 does not involve directly in dHbS disability as discussed in the section "Biophysical interaction-based evaluation" The use of the anti-sickling drug (GBT440) establishes two salt bridges (Table 2) between Glu7 and Lys132 (Fig. 4b), so that the motion of PVG can be stopped, and the HbS structure can be stabilised.Because of this, the conversion of PVG into a moving domain is also stopped.Besides, in GBT440 treated OHbS (5e83 13 ), the additional salt bridge (B:LYS82:NZ-D:HIS146:O) formed between two different β-chains (Table 2) was also found responsible for OHbS stability.It also exhibits how this anti-sickling drug (GBT440) makes OHbS stable by increasing the number of salt bridges.If it is so, all these observed things should be done by another antisickling drug, VZHE-039.When the same things were studied in the case of VZHE-039, it was observed that the VZHE-039 also establishes two salt bridges between the same residues where GBT440 does, but of a longer distance.In VZHE-039 treated HbS, the absence of additional salt bridges and the presence of long-ranged salt bridges indicate the lower ability of VZHE-039 to make the HbS structure stable as compared to GBT-440.www.nature.com/scientificreports/Moreover, the use of the anti-sickling drug (GBT440) also decreases the torsion in the Val6-Glu7 peptide bond to some extent but increases in the Pro5-Val6 peptide bond residue to a greater extent (Fig. 4a).In GBT440 treated OHbS, the Val6-Glu7 peptide torsion was found nearer to the torsion of Glu6-Glu7 peptide in OHbA (Fig. 4a) which evidently shows that the further torsion in Val6-Glu7 peptide to make the Glu7 movable is very difficult.Through this restriction in further torsion at the mutation point (PVG), the structure of OHbS becomes more stable when treated with GBT440.Furthermore, due to this torsional restriction on Val6-Glu7 peptide via GBT440, HbS-PVG attains a similar orientation as HbA-PGG attains, and the Glu7-Lys132 salt bridge distance in OHbS also becomes equal to OHbA (Fig. 4b).Consequently, the conversion of stable OHbS into less stable dHbS is stopped by GBT440.In this way, the anti-sickling drug (GBT440) stops its participation in the polymerization reaction by making the oxygenated form of HbS more stable via additional salt bridges and restriction in PVG peptide bond torsion.Table 3. Dihedral angles involved in Pro-Val-Glu peptide bonds (Fig. 2b) with atoms tagged in HbS (Fig. 2a).C* = Chiral Carbon given in Fig. 2a.www.nature.com/scientificreports/Another reason for the same thing is the hydrophobic nature of Pro-Val-Glu peptides because these peptides have two hydrophobic residues (Pro and Val) and one hydrophilic residue (Glu).On the other hand, Pro-Glu-Glu peptides has two hydrophilic residues (Glu) and one hydrophobic residue (Pro).Actually, the presence of two www.nature.com/scientificreports/hydrophobic amino acids in Pro-Val-Glu peptides them hydrophobic.On the other hand, the binding sites i.e. β88 leucine (Leu-beta88) and β85phenylalanine (Phe-beta85) residues on the adjacent dHbS-M are also hydrophobic 74 .Hence, the attachment of hydrophobic Pro-Val-Glu of HbS with the hydrophobic residues of the adjacent dHbS-M is stronger as compared to that of hydrophilic Pro-Glu-Glu of HbA.It is because hydrophilic compound does not have a greater affinity towards hydrophobic complex, but hydrophobic compound does, as per universal truth.

Key phases before RBC sickling
After a deep computational evaluation of RBC sickling-related data, the steps involved before the polymer formation are summarized below (Fig. 5): (a) Deoxygenation of HbS 5,13 (b) Increase in Glu7-Lys132 salt bridge distance (Fig. 4b) along with the torsional change in peptide bond (Fig. 4a) as per the literature data 7,75 .(c) Destabilization of Glu7-Lys132 salt bridge due to the increased distance 23 (d) Conformational specificity for the fold or function provided by a destabilized salt bridge 14,76 (e) Creation of the moving domain through flexibility 73 caused by hydrophobic nature 14 (f) Insertion of the hydrophobic moving domain into hydrophobic pockets on adjacent dHbS-M 30,34 .(g) Initiation of HbS polymerization for the sickling of RBC.

GBT-440 anti-sickling mechanism
After a deep evaluation of the pdb file with an anti-sickling drug (GBT-440), the mechanism of GBT-440's action to stop the polymerization is summarized below: (a) Accumulation of GBT-440 in oxygenated HbS (b) After accumulation, the condensation reaction between αVal1 N-terminal amines and GBT440 for the formation of Schiff 's base.(c) Variation in torsional strain (Fig. 4a) on peptide bonds after the Schiff 's base formation.(d) The motion of the salt bridge-forming residues toward other ionic residues (Fig. 4a) for torsional strain minimization.(e) Creation of the additional salt bridges (5e83 13 ) in the HbS structure (Table 2) through the motion of ionic residues to each other (Fig. 4a).(f) Establishment of stable OHbS from untreated OHbS due to the formation of additional salt bridges (Fig. 4b) and Table 2. (g) After gaining stability, neither the creation of the hydrophobic moving domain in HbS to be inserted in dHbS-M nor HbS polymerization for RBC sickling.
The effect of carbon monoxide combination with HbA was observed by studying the variation in interactions among polypeptide residues.The increased number of salt bridges in HbA the addition of CO openly indicates its stability.Hence, HbA gets stability through the increased number in the salt bridge after combination www.nature.com/scientificreports/with CO, while no salt bridge increment was observed in HbA oxygenated with oxygen gas (Table 1).Due to this, HbA combines with CO more tightly as compared to O 2 .Besides, no changes occur in the structure of CO, but that in HbA occurs after a combination of both.Therefore, in HbA and CO combination, HbA has a greater affinity for the same, but CO does not.
During the study of the pH effect on dHbS polymerization, it was found that the protonation of only Lys132 was just because of the lone pair donor atom in Lys132 i.e. nitrogen atom of the amine group (-NH 2 ) of Lys132 (Fig. 6).The nitrogen atom of the amine group (-NH 2 ) makes a coordinate bond with the H + ion to convert into its protonated form (-NH 3 + ).On the other hand, the deprotonated form of Glu7 has a carboxylate ion (Fig. 6) which is considered more stable due to the presence of delocalized electrons 77 .Hence, the protonation of Glu7 does not happen to maintain the stability of Glu7.Moreover, the decreased salt bridge distance was just because of the existence of H + ion between Glu7 and Lys132 due to which Glu7 becomes unable to make a salt bridge with Lys132 directly (Fig. 6).In this way, the ionic strength of Lys132 residue to make HbS structure stable is diminished because the protonation of the polymeric chain makes the ionic strength weaker 78 .In the same way, the protonation also makes the polypeptide chain weaker because as per literature data 78 , the protonation involves breaking intra-chain hydrogen bonds.Based on this information, it can be pointed out that not only the dHbS instability but also the instability of the Glu7-Lys132 salt bridge makes the local environment flexible.This is because the destabilizing salt bridges make local regions flexible for better fit due to functional requirements 20 .Notably, the effective folding rates of the Lys-based peptides at acidic pH are slightly slower 75 which also shows that the unfolding in the HbS protein chain becomes very easy for the conversion of a peptide chain into a moving domain.
Additionally, when the entry effect of 2,3-DPG and GBT440 in the hemoglobin structure was analyzed, it was noted that the effect of 2,3-DPG entry in hemoglobin structure (1b86 72 ) was found just conflict with that of GBT440 (5e83 13 ).The 2,3-DPG makes a salt bridge (Fig. 7a,b) with 'Lys residue' of ASN-LEU-LYS-GLY-THR peptides present in the hemoglobin β-chain (1b86 72 ).Interestingly, the same 'Lys residue' was found attached to the 'His residue' via an additional salt bridge (B:LYS82:NZ-D:HIS146:O) (5e83 13 ) in the GBT440-treated HbS structure.Hence, it can be concluded that the presence of GBT440 allows the 'Lys residue' to make the salt bridge with the 'His residue' , but that of 2,3-DPG does not.Moreover, the most important fact regarding the additional salt bridge (B:LYS82:NZ-D:HIS146:O) in GBT440-treated HbS, was its existence between two different β-chains (B and D) because both residues were found on different β-chains (5e83 13 ).It confirms the stability of GBT440treated HbS in which the two different β-chains are connected via B:LYS82:NZ-D:HIS146:O in which the 'Lys residue' of the 'B chain' interacts with another ionic residue of the 'D chain' (Table 2).Therefore, it is clear that the GBT440 increases the number of salt bridges in HbS (Fig. 4b) to make the HbS protein more stable.On the other hand, the binding of 2,3-DPG in the β-cleft promotes the polymerization of deoxy-HbS 10 .It is because the 2,3-DPG does not let the Lys residue of ASN-LEU-LYS-GLY-THR peptides to make a salt bridge with 'His residue' as GBT440 does.Due to this, the hemoglobin structure with 2,3-DPG becomes less stable.In this way, we can say that the 2,3-DPG creates a disturbance via salt bridge formation in the hemoglobin structure and makes it less stable.This destabilization is managed in HbA properly 42 but not in HbS as per the presented study.Hence, the 2,3-DPG entry in HbA is not found as dangerous as in HbS.
The entry/removal of 2,3-DPG is associated with pH variation.The low pH is responsible for the removal of 2,3-DPG, while the higher pH is responsible for the entry of 2,3-DPG as per the introductory part.Actually, it is just because of the presence of two phosphate (PO 4 (Fig. 7a,b) which are less stable but strong bases as they are produced from their respective stable acids i.e. phosphoric acid (H 3 PO 4 ) and carboxylic acid (-COOH).It can be explained by keeping the facts collected after getting literature data.For instance, we know that the phosphoric acid (H 3 PO 4 ) is a weak and stable acid 79,80 , so the phosphate ion (PO 4 3− ) must be a strong base with less stability according to the collected works (https:// www.chem.fsu.edu/ cheml ab/ Maste ring/ Phosp hateB uffers.htm) because a base of a chemical species will be strong if its protonated form is classified as a weak 81 .Therefore, the conversion of phosphoric acid to phosphate is a very slow process but that of phosphate to phosphoric acid is very fast (Fig. 7c).Consequently, the affinity of less stable phosphate ion (PO 4 3− ) for H + or protonation is found more to get stabilized form.Besides, the presence of two phosphate (PO 4 3− ) ions in the 2,3-DPG structure (Fig. 7a) is also found responsible for the formation a salt bridge with 'Lys residue' .It is because the salt bridge is formed between two oppositely charged species 21 for which the phosphate group (PO 4 3− ) has a negative charge while 'Lys residue' has a positive charge.On decreasing pH or increasing H + ion concentration, the protonation of two negatively charged phosphate (PO 4 3− ) ions takes place (Fig. 7d).Such type of protonation neutralizes the negatively charged phosphate 82 to convert into their protonated and neutral form (Fig. 7d).In this way, after loosing its negative charge, the 2,3-DPG becomes unable to make the salt bridge with positively charged 'Lys residue' , and conditions favors the removal of 2,3-DPG from HbS structure.Hence, the deprotonated 2,3-DPG conversion into protonated 2,3-DPG was found responsible for not only 2,3-DPG less stability but also its removal from the intracellular environment of low pH (Fig. 7d).Among all these described things, the involvement of phosphatase activation at low pH can not be ignored.Phosphatase removes the phosphate group from the organic residue 83 .The activation of phosphatase is also found responsible for the removal of 2,3-DPG from intracellular medium 84 .The medium of the intracellular environment is made acidic by the addition of CO 2. It is because carbon dioxide, already acidic in nature 85 , forms carbonic acid (Fig. 7d) after the reaction with water [86][87][88] .Hence, the higher concentration of CO 2 means more production of carbonic acid which makes the intracellular medium acidic by lowering pH (7-5.6) 89.Along with lowering the pH value, the phosphatase becomes active since the phosphatase is inactive at pH levels above 7.2 but is more active at the lower pH in RBCs 90 .Hence, after activation at low pH, the phosphatase starts to cleave the ester bond formed between the phosphate group and organic residue i.e. glyceric acid (Fig. 7d).At low pH, the cleavage of phosphate groups becomes easier due to the lack of 2,3-DPG connection with 'Lys residue' as described above.In this way, the phosphatase activation contributes to the decreased 2,3-DPG levels 90 by converting it into 3-phosphoglycerate 91,92 (Fig. 7d).Therefore, in the presented study, the lower pH was found responsible for not only the weaker/unstable 2,3-DPG connection with 'Lys residue' but also the phosphatase www.nature.com/scientificreports/activation.On the other hand, all these things happen oppositely at higher pH which was found responsible for not only 2,3-DPG stability but also its entry in the intracellular environment.Henceforward, the same mechanism of 2,3-DPG to cause trouble in hemoglobin can be applied in the HbStyped hemoglobin before polymerization.It is because the effect created by the entry of 2,3-DPG in normal hemoglobin was found very adverse in sickle hemoglobin as the 2,3-DPG entry helps in the oxygen release from HbA 93 i.e. deoxygenation of HbA.The deoxygenated form of HbA can get stabilized but that of HbS is not, as described above, that is why dHbS undergoes polymerization.Keeping the described facts into consideration, the depletion of 2,3-DPG could also play a key role in the control of HbS polymerization, which has been suggested recently 44 .
After a deep evaluation of RBC sickling-based literature data, a symbiotic effect of pH and 2,3-DPG on the dHbS solubility was observed.Besides, the pH effect seemed just like a mysterious fact that was found associated with dHbS polymerization before RBCs sickling.Truly, RBC sickling can occur in both conditions i.e. whether the pH of the intercellular medium is increased 39 or decreased 39,95 .At higher pH (more than 7.4), the HbS polymerization occurs due to the decreased solubility caused by 2,3-DPG accumulation 39 (Fig. 8), while at lower pH (less than 7.25), the HbS polymerization can also take place just because of HbS gelation 39 (Fig. 8).It is because the increase in pH promotes the accumulation of 2,3-DPG that causes a large release of oxygen from red cells, whose risk of sickling is high 39 .Moreover, the intracellular pH was found 7.41 in the case of HbS deoxygenation 10 .It shows that the higher pH value of the intracellular medium favors the release of oxygen from HbS caused by 2,3-DPG accumulation.On the other hand, the decrease in pH causes gelation which is also found responsible for a large release of oxygen from red cells, whose risk of sickling is high 39 .Moreover, the intracellular pH was found 7.13 in the case of HbS oxygenation 10 .At this pH, the HbS gelation was confirmed when a sharp increase in the gelling tendency was observed below the pH 7.15 39,94 .We know that gelation is a general way to convert a fluid into a solid 96 .Hence, the gelation increases the density of a substance 96 .The same things can be applied to the HbS gelation at low pH, which occurs due to the protonation of the HbS polypeptide chain because the protonation not only makes a polymeric chain denser but also provides stiffness to it 78 .It has already been confirmed that denser HbS cells also cause low oxygen affinity, and increase the risk of RBC sickling 97 .Moreover, the protonation diminishes the ionic strength of a polymeric chain 78 ; which clearly shows the HbS disability via weak ion strength (salt bridge) at low pH.In this way, we can say that the protonation of the HbS polypeptide chain occurs in an acidic medium continuously until its ionic strength diminishes.The completion of protonation of the HbS polypeptide chain may occur at pH 7.15 after which HbS gelation starts 39 , and then polymerization occurs for RBC sickling.It is because no pH effect was observed on Hb and Hb-DPG solubility 98 below 7.15 pH or neutral pH (Fig. 8) i.e. no more consumption of proton (H + ) as all residues get protonated completely at pH 7.15.The same results were observed below pH 7 when only a slight drop in the maximum number of sickled cells 39 was noted.It openly revealed no need for protonation as all residues get protonated at pH 7.15.

Conclusion
Based on the interpretations of the results, it can be concluded that many factors were found associated with HbS polymerization after deoxygenation.In these factors, peptide bond torsion, salt bridge number, salt bridge strength, and hydrophobicity can be included.In this way, we can say that an increase in the HbS oxygen affinity; HbS solubility; and HbS stability through the increased salt bridge number could stop HbS polymerization easily.In addition, the intracellular elimination of 2,3-DPG and pH maintenance can also do the same thing.Of course, the separate effects of 2,3-diphosphoglycerate and intracellular pH on HbS were also observed, but they were found to show symbiotic effects on the dHbS solubility at equilibrium that were shown responsible for HbS polymerization in RBCs.
Moreover, the mechanism and strength of the antisickling drugs other than GBT440 can also be evaluated by following the presented method.It can also be concluded that the antisickling drug that forms the Schiff base with 'Val residue' of α-chain terminal would behave like GBT440 or not, is not confirmed.For the evaluation of its antisickling properties, we have to study its accumulation effect in the HbS structure including the salt bridge number, peptide bond torsion, and other factors as described above.By doing so, it would become very easy to know how the newly synthesized drug during its antisickling activity makes the HbS structure not only stable but also unconvertable in the moving domain.

Simulation study
In the presented computational study, the pdb files of oxygenated normal hemoglobin (OHbA), deoxygenated normal hemoglobin (dHbA), oxygenated sickle hemoglobin (OHbS), and deoxygenated sickle hemoglobin (dHbS) with pdb codes 1hho 67 , 1a3n 68 , 5e6e 69 and 2hbs 70 respectively, were obtained from the protein data bank.After that, all significant physiochemical parameters of all obtained files were studied and compared deeply.Besides the taken pdb files [67][68][69][70] , the most significant data of other pdb files such as 5e83 13 , 6xd9 53 , 2m6z 71 , and 1b86 72 was also included in the current study.The pdb file with pdb code 5e83 13 was taken for the structural study of OHbS having an anti-sickling drug, GBT440 so that its mechanism to make the HbS structure stable and to stop the HbS polymerization, could be explained.On the other hand, a 6xd9 53 pdb coded file was used for a comparative study purpose so that the comparison between GBT440 and VZHE-039 could be done to evaluate their abilities to make the HbS structure stable.It is because the drug named VZHE-039 has also been suggested as a candidate to stop RBCs sickling.The pdb file with pdb code 2m6z 71 was taken to know the effect of carbon monoxide on HbS, while the effect of 2,3-diphosphoglycerate (2,3-DPG) entry in hemoglobin structure was analyzed by the evaluation of pdb file with pdb code 1b86 72 .The main purpose of the presented computational study was to answer of different aspects mentioned in the introductory part through the deep analysis of all Hbrelated structures.Some of them are as follows:

Physio-chemical interaction study
For the study of physio-chemical interaction-based differences among biological molecules (OHbA, dHbA, OHbS, and dHbS), all their pdb files were opened in Discovery Studio 2019, and all types of physio-chemical interactions such as salt bridge (Fig. 9), hydrogen bond, and charge-dipole interaction were noted and used for the result interpretation.The salt bridge interactions are relatively strong non-bonded interactions between pairs of oppositely charged groups where hydrogen bonding also occurs.To know the mechanism of the anti-sickling drug action, the analysis of electrostatic interactions, especially salt bridges number in GBT440 treated OHbS (5e83 13 ) was done by comparing its β-chain with those of OHbA, dHbA, OHbS, and dHbS having pdb codes: 1hho 67 , 1a3n 68 , 5e6e 69 and 2hbs 70 respectively.
Besides, the peptide bond torsion was also studied among the same.It is because amino acids in a protein chain feel torsion strain [101][102][103] which can cause a resistance to bond rotation and can influence a barrier to rotation 103 .Hence, the Ramachandran plot-based study was also done to evaluate the change in peptide bond torsion occurring in Pro-Glu-Glu as well as Pro-Val-Glu along with the OHbA/OHbS conversion into dHbA/ dHbS.The inclusion of the Ramachandran plot-based observations was found very supportive in the result interpretation of the presented study.All these similar things were done with GBT440-containing OHbS to know how this anti-sickling drug affects the peptide bond torsion in HbS structure, and how it makes the behavior of OHbS equal to OHbA.In the study of physiochemical differences among biological molecules (OHbA, dHbA, OHbS, and dHbS), the result interpretations gave many key points.Based on these significant points, it became very easy to explain the reason for dHbS polymerization before RBCs sickling.The additional literature data related to the taken biological molecules was also included in the result interpretation to support the main find findings of the physiochemical study.The main purpose of the presented study was to evaluate different parameters changed during the OHbS conversion to dHbS.Here, the scanty data related to the changes occurring in HbA during the same were also evaluated for a comparative study between HbA and HbS.Due to this, the torsion angles associated with Pro-Val and Val-Glu peptide bonds before and after deoxygenation in the HbS protein were studied genuinely but not in the HbA protein.For this purpose, phi (φ) was the Ca(i-1), C(i), N(i), Ca(i) torsion angle in the Pro-Val peptide bond in which Cα (i-1), C(i), N(i), Cα(i) were tagged as C9, N15, C10, C16 respectively (Fig. 2a,b), while psi (ψ) was the Ca(i), C(i), N(i + 1), Ca(i + 1) torsion angle in Val-Glu in which Cα(i), C(i), N(i + 1), Cα(i + 1) were tagged as C16, C17, N22, C23 (Fig. 2a,b).An amino acid needs to be bonded to two other amino acids to have values set for both psi and phi, for that reason, the two terminal amino acids (Thr and Lys) do not have values set for these angles.

The study of pH, CO, CO 2, and 2,3-DPG effects on HbS
Of course, many studies 39 have shown the dependency of dHbS polymerization on pH.As per the introductory part of the presented article 7,39 , the dHbS polymerization is accelerated by an acidic medium.It is well understood that the acidic medium has a large number of H + ions.Due to this, many residues present in the peptide chain get protonated.In the presented study, only salt bridge-forming residues present in PVG and PGG were protonated computationally i.e.Glu7.Besides Glu7, the residue 'Lys132' was also protonated as it was found attached to Glu7 via salt bridge formation.It is because the salt bridge is related to not only the stability of the peptide chain [19][20][21] but also the folding of a polypeptide chain 36,37 .In the presented work, after getting the protonated form of the salt-forming residues, the salt bridge strength with respect to its connection between Glu7 and Lys132 was studied so that the acidic medium-based facts could be evaluated deeply.In the same way, the effect of CO 2 in HbS structure was studied computationally, while the effect of 2,3-DPG on HbS physiochemical parameters in pdb files with PDB Codes 1b86 72 was also studied.The PDB file with pdb code 2m6z 71 was evaluated to know the effect of CO.It is because the hemoglobin structure gets stability after combining with CO which is why it combines with CO more tightly but with O 2 , loosely 104 .After combination, the interaction-based stability caused by CO addition in hemoglobin was not found as satisfactory as expected.Hence, the reason for the interactionbased stability of hemoglobin caused by CO is yet to be explained.

Figure 2 .
Figure 2. (a) Atoms tagged for torsion angle study in HbS, and (b) torsion angles designed for Pro-Val and Val-Glu peptide bond in (a).

Figure 4 .
Figure 4. (a) OHbS → dHbS conversion involving the distortion in PVG residues torsion, (b) biophysical interactions made by PGG/PVG residues of HbA/HbS in different conditions, and (c) flexibility found in the hydrophobic domain 73 .

Figure 8 .
Figure 8.The pH effect on the solubility, gelation, binding affinity for oxygen, and polymerization of HbS; (a) 66,106 .

Table 1 .
Physiochemical interactions involved in PGG of HbA.*Oxygenated with CO.

Table 2 .
Physicochemical interactions involved in PVG of HbS.*Oxygenated with CO; **Additional salt bridge.

Table 4 .
Peptide bond torsion in PGG and PVG residues.